The Effects Of Bisoprolol On Hypoxia/Reoxygenation-induced Apoptosis In H9c2 Cells And Its Mechanisms

Background:The reperfusion therapy is the effective treatment of acute myocardial infarction (AMI), and with the rapid development of coronary artery bypass grafting (CABG) and the coronary artery forming (PTCA) and revascularization, AMI reperfusion therapy appeared a leap. However, the occlusion artery reperfusion itself can cause myocardial cell damage, which called myocardial ischemia reperfusion injury (MIRI). In the process of MIRI, reperfusion injury salvage kinase signaling pathway (RISK) are activated, including phosphatidylinositol 3-kinase (PI3K), protein kinase B (PKB, also known as Akt), endothelial nitric oxide synthase (eNOS), ERK1/ 2, PKC, JAK-STAT. PI3K/AKT and its downstream GSK3β signaling pathway,which is an important and protective pathway of reperfusion. During the process of ischemia and reperfusion, a great number of (3 receptors activated on the surface of cardiomyocytes, especially, the activation of β1 receptors can increase the ROS production and cell apoptosis of cardiomyocytes. Bisoprolol, a high selective β1 blocker, could decrease sympathetic activity after ischemia reperfusion, and play a role of the antioxidant and resistance to apoptosis. Based on this, we establish cardiomyocytes model of hypoxia/reoxygenation to simulate myocardial ischemia reperfusion injury, and investigate the effect of bisoprolol on hypoxia /reoxygenation-induced apoptosis of H9c2 cardiomyocytes, we also use blockers LY294002 and siRNA interference to study the role of PI3K/AKT/GSK-3β pathway involvedObjective:To investigate the effect and mechanisms of bisoprolol on H/R induced apoptosis of H9c2 cardiomyocytes.Methods:Part 1 Study of the effect of bisoprolol on H/R induced apoptosis of H9c2 cells.Cultured H9c2 cells were divided into 3 groups before subject to ischemia/reperfusion injury:normal group (control); hypoxia/reoxygenation group(H/R); hypoxia/reoxygenation+bisoprolol group(H/R+B). H/R group was treatedwith 6 hours hypoxia and 2 hours reoxygenation. The survival of the cells was measured by MTT, cell apoptosis and radical oxygen species (ROS) rates were evaluated by flow cytometry. The protein level of phosphyorylated AKT and phosphyorylated GSK3β was determined by Western Blotting.Part 2 Uitilizing LY294002 decreased the expression of Akt and siRNA silencing GSK-3(3 to study whether its mechanism was involved in the activation of PI3K/AKT/Gsk-3βpathway.H9c2 cells were divided into 2 groups before subject to stimulated myocardial ischemia/reperfusion injury:(1) the LY294002 treatment:hypoxia/reoxygenation group(H/R); hypoxia/reoxygenation+bisoprolol group(H/R+B); hypoxia/ reoxygenation+bisoprolol+LY294002 group(H/R+B+LY).(2) the GSK3P siRNA silencing:control group (control group), hypoxia/reoxygenation (H/R), hypoxia/ reoxygenation+bisoprolol group(H/R+B), siRNA group (siRNA), siRNA+ hypoxia/reoxygenation group (siRNA+H/R), siRNA+ hypoxia/reoxygenation+bisoprolol group (siRNA+H/R+B). The viability of myocardial cells was detected by MTT method. Apoptosis and ROS were assayed by flow cytometry. The expression of p-P38 and p-AKT proteins were also investigated by Western Western blot.Results:Part 11. Bisoprolol enhanced cell viability of H9c2 cardiomyocytes subjected to H/R.2. Bisoprolol reduced apoptosis of H9c2 cardiomyocytes subjected to H/R.3. Bisoprolol alleviated intracellular ROS of H9c2 cardiomyocytes subjected to H/R.4. Bisoprolol upregulated phosphorylation of AKT and GSK-3β of H9c2 cardiomyocytes subjected to H/R.Part 21. The effects of bisoprolol on protection H9c2 cardiomyocytes including cell viability, apoptosis, ROS, were inversed by LY294002.2. The effect of bisoprolol upregulating phosphorylation of AKT and GSK-3β of H9c2 cardiomyocytes subjected to H/R was partly negated by LY294002.3. The effects on Cell viability cell apoptosis and intracellular ROS by using bisoprolol in H9c2 were suppressed by si-RNA interference.4. The effect of bisoprolol upregulating phosphorylation of AKT and GSK-3β of H9c2 cardiomyocytes subjected to H/R was partly negated by siRNA interference.Conclusion:Bisoprolol can protect the cardiomyocytes from hypoxia/reoxygenation induced injury. Its mechanism is involved in the activation of PI3K/AKT/Gsk-3β survival pathway via increasing the phosphorylation of AKT and GSK-3β,while reducing the production of ROS and increasing cell survival.